MCU Upregulation Overactivates Mitophagy by Promoting VDAC1 Dimerization and Ubiquitination in the Hepatotoxicity of Cadmium

Abstract Cadmium (Cd) is a high‐risk pathogenic toxin for hepatic diseases. Excessive mitophagy is a hallmark in Cd‐induced hepatotoxicity. However, the underlying mechanism remains obscure. Mitochondrial calcium uniporter (MCU) is a key regulator for mitochondrial and cellular homeostasis. Here, Cd exposure upregulated MCU expression and increased mitochondrial Ca2+ uptake are found. MCU inhibition through siRNA or by Ru360 significantly attenuates Cd‐induced excessive mitophagy, thereby rescues mitochondrial dysfunction and increases hepatocyte viability. Heterozygous MCU knockout mice exhibit improved liver function, ameliorated pathological damage, less mitochondrial fragmentation, and mitophagy after Cd exposure. Mechanistically, Cd upregulates MCU expression through phosphorylation activation of cAMP‐response element binding protein at Ser133(CREBS133) and subsequent binding of MCU promoter at the TGAGGTCT, ACGTCA, and CTCCGTGATGTA regions, leading to increased MCU gene transcription. The upregulated MCU intensively interacts with voltage‐dependent anion‐selective channel protein 1 (VDAC1), enhances its dimerization and ubiquitination, resulting in excessive mitophagy. This study reveals a novel mechanism, through which Cd upregulates MCU to enhance mitophagy and hepatotoxicity.

negative control (NC) siRNA and three different designed siRNAs for 48 hours, respectively.
The bold font indicated the most effective siRNA that was used in this study. As to MCU, CREB, CypD and VDAC1, we further verified the knockdown effect of selected siRNA with three replicates. I) Immunoblots for HA-VDAC1 in lysates from cells infected with lentivirus containing exogenous VDAC1 gene fused with HA-tag. The molecular weight was shown as indicated. J) Immunofluorescence staining of HA-VDAC1 in cells infected with HA-VDAC1 lentivirus or not, and the images were taken under inverted fluorescence microscope. Scale bar, 200 μm. Indicated primary and secondary antibodies were used in these immunoblots.   (n = 20). C) Cell viability after 3-MA treatment in Cd-exposed cells (n = 5). D) Analysis of apoptosis and necrosis in HepG2 cells by Hoechst 33342 (blue) and propidium iodide (PI, red) stain. 100 × magnification images were taken and representative results were shown. Scale bar, 100 μm. E) Apoptosis rate and F) necrosis rate were calculated from six 100 × magnification images. Normal cells showed as weak blue and red signal. Apoptotic cells showed as bright blue and weak red signal. Necrotic cells showed as bright blue and red signals. **p < 0.01, ***p < 0.001. ns, no significance.

Figure S5
Supplementary figure 5: Effects of Baf A1 on Cd-induced hepatotoxicity. A) Confocal images taken from HepG2 cells transfected with EGFP-mCherry-LC3 adenovirus 24 hours before Baf A1 pretreatment and Cd exposure. Yellow dot indicated the autophagosome, and free red dot indicated the autolysosome. Scale bar, 5 μm. B) Quantification of autophagosomes and autolysosomes from (A), n = 20. C) Cell viability after Baf A1 treatment in Cd-treated cells (n = 5). D to F) Apoptosis and necrosis analysis as described above in S2 after Baf A1 treatment. **p < 0.01, ***p < 0.001. ns, no significance.

Figure S6
Supplementary figure 6: Gene and protein expression levels for MCU subunits. A) Gene expression levels of MCU, EMRE, MCUb, MICU1 and MICU2 after Cd exposure. The cells were exposed with or without 12 μM Cd for 12 hours, and the total RNA were harvested according to the requirments of RNA-seq (n = 4). B) Immunoblots for EMRE, MCUb, MICU1 and MICU2 in cells treated with or without 3, 6 and 12 μM Cd for 12 hours. GAPDH was used as the loading control. C) Quantitative analysis of (B). *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance. Two pairs of primers, FR and FV, were designed for mcu genotyping. Mice tail was cut for harvest of DNA, followed by PCR and agarose gel electrophoresis. The WT, HZ or KO genotype was confirmed by existence of single FR signal (~300bp), both FR and FV signal (~200bp), and single FV signal, respectively. C) Immunoblots for MCU in liver lysates from WT, HZ and KO mice. D) Body weight of WT, HZ and KO mice at 6 th week in normal conditions (n = 10). E) Body weight increment of WT or HZ mice treated with saline or Cd (n = 10). ***p < 0.001. ns, no significance.  after siMCU and Ru360 treatment, respectively. B, E) Apoptotic rate was calculated from six 100 × magnification images after siMCU and Ru360 treatment, respectively. C, F) Necrotic rate was also calculated from six 100 × magnification images after siMCU and Ru360 treatment, respectively. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.

Figure S12
Supplementary figure 12: Cd promoted opening of mitochondrial permeability transition pore. A) mPTP assay kit was used to evaluate the opening of mPTP. CAM (calcein acetoxymethyl ester) could penetrate into cytoplasm and other subcellular organelles (mainly mitochondria) and be hydrolyzed to calcein. Calcein showed bright green fluorescence that could be quenched by CoCl2. The more mPTP open, the lower mitochondrial fluorescence intensity. Cells were exposed to 3, 6 and 12 μM Cd for 6 hours, and the fluorescence was detected and quantified after CAM and CoCl2 addition (n = 5). B) Mitochondrial membrane potential evaluated by TMRM. Cells were exposed to 3, 6 and 12 μM Cd for 12 hours and the fluorescence intensity was detected by microplate reader at 550/575 nm (n = 5). C) Evaluation of mitochondrial morphology after 3, 6 and 12 μM Cd exposure for 12 hours. Mitochondria were stained by MitoTracker Red (200 nM).